Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry

A highly selective, amiloride-sensitive, epithelial sodium channel from rat colon (rENaC), composed of three homologous subunits termed alpha, beta, and gamma rENaC, has been cloned by functional expression and was proposed to mediate electrogenic sodium reabsorption in aldosterone- responsive epithelia. To determine whether rENaC could account for sodium absorption in vivo, we studied the cellular localization of the sodium channel messenger RNA subunits by in situ hybridization and their cellular and subcellular distribution by immunocytochemistry in the kidney, colon, salivary, and sweat glands of the rat. In the kidney, we show that the three subunit mRNAs are specifically co- expressed in the renal distal convoluted tubules (DCT), connecting tubules (CNT), cortical collecting ducts (CCD), and outer medullary collecting ducts (OMCD), but not in the inner medullary collecting ducts (IMCD). We demonstrate co-localization of alpha, beta, and gamma subunit proteins in the apical membrane of a majority of cells of CCD and OMCD. Our data indicate that alpha, beta, and gamma subunit mRNAs and proteins are co-expressed in the distal nephron (excepting IMCD), a localization that correlates with the previously described physiological expression of amiloride-sensitive electrogenic sodium transport. Our data, however, suggest that another sodium transport protein mediates electrogenic amiloride-sensitive sodium reabsorption in IMCD. We also localized rENaC to the surface epithelial cells of the distal colon and to the secretory ducts of the salivary gland and sweat gland, providing further evidence consistent with the hypothesis that the highly selective, amiloride-sensitive sodium channel is physiologically expressed in aldosterone-responsive cells.     

An Alu cassette in the human epithelial sodium channel

Here, we report the presence of two splice variants of the human epithelial sodium channel alpha subunit (h alpha ENaC) containing Alu cassette, namely h alpha ENaC+22 and h alpha ENaC+Alu, in various tissues. Functional expression of these splice variants with hENaC beta and gamma subunits produced loss-of-channel activity in the Xenopus oocyte expression system. Interestingly, coexpression of h alpha ENaC+22 or h alpha ENaC+Alu, respectively, with wild type hENaC alpha, beta, and gamma subunits enhanced the expression of amiloride-sensitive current in oocytes. The presence of Alu sequences in the 3'-untranslated region of h gamma ENaC was also identified.     

Identification of a PY motif in the epithelial Na channel subunits as a target sequence for mutations causing channel activation found in Liddle syndrome.

Liddle syndrome is an autosomal dominant form of hypertension, resulting from mutations in the cytoplasmic C-terminus of either the beta or gamma subunits of the amiloride-sensitive epithelial Na channel (ENaC) which lead to constitutively increased channel activity. Most mutations reported to date result in the elimination of 45-75 normal amino acids from these segments, leaving open the question of the identity of the precise amino acids in which mutation can lead to an enhanced channel activity. To address this question, we have performed a systematic mutagenesis study of the C-termini of the alpha, beta and gamma ENaC subunits of the rat channel and have analyzed their function by expression in Xenopus oocytes. The results demonstrate that a short proline-rich segment present in the cytoplasmic C-terminus of each subunit is required for the normal regulation of channel activity. Missense mutations altering a consensus PPPXY sequence of the alpha, beta or gamma subunits reproduced the increase in channel activity found in mutants in which the entire cytoplasmic C-termini are deleted. This proline-rich sequence, referred to as the PY motif, is known to be a site of binding by proteins bearing a WW domain. These findings show that the three PY motifs in the C-termini of ENaC are involved in the regulation of channel activity, probably via protein-protein interactions. This new regulatory mechanism of channel function is critical for the maintenance of normal Na reabsorption in the kidney and of Na+ balance and blood pressure.     

The heterotetrameric architecture of the epithelial sodium channel (ENaC).

The epithelial sodium channel (ENaC) is a key element for the maintenance of sodium balance and the regulation of blood pressure. Three homologous ENaC subunits (alpha, beta and gamma) assemble to form a highly Na+-selective channel. However, the subunit stoichiometry of ENaC has not yet been solved. Quantitative analysis of cell surface expression of ENaC alpha, beta and gamma subunits shows that they assemble according to a fixed stoichiometry, with alpha ENaC as the most abundant subunit. Functional assays based on differential sensitivities to channel blockers elicited by mutations tagging each alpha, beta and gamma subunit are consistent with a four subunit stoichiometry composed of two alpha, one beta and one gamma. Expression of concatameric cDNA constructs made of different combinations of ENaC subunits confirmed the four subunit channel stoichiometry and showed that the arrangement of the subunits around the channel pore consists of two alpha subunits separated by beta and gamma subunits.     

Kainic acid-induced status epilepticus alters GABA receptor subunit mRNA and protein expression in the developing rat hippocampus

Kainic acid-induced status epilepticus leads to structural and functional changes in inhibitory GABAA receptors in the adult rat hippocampus, but whether similar changes occur in the developing rat is not known. We have used in situ hybridization to study status epilepticus-induced changes in the GABAAalpha1-alpha5, beta1-beta3, gamma1 and gamma2 subunit mRNA expression in the hippocampus of 9-day-old rats during 1 week after the treatment. Immunocytochemistry was applied to detect the alpha1, alpha2 and beta3 subunit proteins in the control and treated rats. In the saline-injected control rats, the alpha1 and alpha4 subunit mRNA expression significantly increased between the postnatal days 9-16, whereas those of alpha2, beta3 and gamma2 subunits decreased. The normal developmental changes in the expression of alpha1, alpha2, beta3 and gamma2 subunit mRNAs were altered after the treatment. The immunostainings with antibodies to alpha1, alpha2 and beta3 subunits confirmed the in situ hybridization findings. No neuronal death was detected in any hippocampal subregion in the treated rats. Our results show that status epilepticus disturbs the normal developmental expression pattern of GABAA receptor subunit in the rat hippocampus during the sensitive postnatal period of brain development. These perturbations could result in altered functional and pharmacological properties of GABAA receptors.     

Preferential assembly of epithelial sodium channel (ENaC) subunits in Xenopus oocytes: role of furin-mediated endogenous proteolysis

The epithelial sodium channel (ENaC) is preferentially assembled into heteromeric alphabetagamma complexes. The alpha and gamma (not beta) subunits undergo proteolytic cleavage by endogenous furin-like activity correlating with increased ENaC function. We identified full-length subunits and their fragments at the cell surface, as well as in the intracellular pool, for all homo- and heteromeric combinations (alpha, beta, gamma, alphabeta, alphagamma, betagamma, and alphabetagamma). We assayed corresponding channel function as amiloride-sensitive sodium transport (I(Na)). We varied furin-mediated proteolysis by mutating the P1 site in alpha and/or gamma subunit furin consensus cleavage sites (alpha(mut) and gamma(mut)). Our findings were as follows. (i) The beta subunit alone is not transported to the cell surface nor cleaved upon assembly with the alpha and/or gamma subunits. (ii) The alpha subunit alone (or in combination with beta and/or gamma) is efficiently transported to the cell surface; a surface-expressed 65-kDa alpha ENaC fragment is undetected in alpha(mut)betagamma, and I(Na) is decreased by 60%. (iii) The gamma subunit alone does not appear at the cell surface; gamma co-expressed with alpha reaches the surface but is not detectably cleaved; and gamma in alphabetagamma complexes appears mainly as a 76-kDa species in the surface pool. Although basal I(Na) of alphabetagamma(mut) was similar to alphabetagamma, gamma(mut) was not detectably cleaved at the cell surface. Thus, furin-mediated cleavage is not essential for participation of alpha and gamma in alphabetagamma heteromers. Basal I(Na) is reduced by preventing furin-mediated cleavage of the alpha, but not gamma, subunits. Residual current in the absence of furin-mediated proteolysis may be due to non-furin endogenous proteases.     

All three WW domains of murine Nedd4 are involved in the regulation of epithelial sodium channels by intracellular Na+

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of alpha, beta, and gamma subunits. The carboxyl terminus of each ENaC subunit contains a PPxY motif which is necessary for interaction with the WW domains of the ubiquitin-protein ligase, Nedd4. Disruption of this interaction, as in Liddle's syndrome where mutations delete or alter the PY motif of either the beta or gamma subunits, results in increased ENaC activity. We have recently shown using the whole-cell patch clamp technique that Nedd4 mediates the ubiquitin-dependent down-regulation of Na+ channel activity in response to increased intracellular Na+. In this paper, we demonstrate that WW domains 2 and 3 bind alpha-, beta-, and gamma-ENaC with varying degrees of affinity, whereas WW domain 1 does not bind to any of the subunits. We further show using whole-cell patch clamp techniques that Nedd4-mediated down-regulation of ENaC in mouse mandibular duct cells involves binding of the WW domains of Nedd4 to three distinct sites. We propose that Nedd4-mediated down-regulation of Na+ channels involves the binding of WW domains 2 and 3 to the Na+ channel and of WW domain 1 to an unknown associated protein.     

Preferential reduction of Na+/K+ ATPase alpha3 by 17beta-estradiol influences contraction frequency in rat uteri

One beta1 and two alpha (alpha1 and alpha3) isoforms of Na+/K+-ATPase exist in rat uteri. Previous immunocytochemistry studies have suggested that the alpha3 isoform may be involved in calcium regulation indirectly. Estrogens are known to both modulate Na+/K+-ATPase activities in non-uterine tissues and suppress spontaneous uterine contractions in rats. Thus the purpose of this study was to examine the correlation between estrogens-modulated uterine contraction and the expression of Na+/K+-ATPase alpha3 isoform in rats. After 1-, 2-, and 4- day treatments with 17beta-estradiol (E2, 5 microg/ml/kg, s.c., daily), the diameter of uterine horn was measured. The contraction force of uterine strips was measured by standard muscle bath apparatus. The protein abundance and enzyme activity of Na+/K+-ATPase in rat uteri were measured by Western blot analysis and ATPase assay, respectively. One day of E2 decreased both contraction frequency and alpha3-protein expression without the change in uterine diameter, enzyme activity or other isoforms. Two days of E2 reduced contraction frequency, the enzyme activity, as well as alpha3- and beta1- protein abundance but increased alpha1-protein and uterine diameter. Four days of E2 elicited similar effects as two days of E2, but did not affect alpha1-protein abundance. In conclusion, E2 elicits differential effects on isoform expression. After 1-day treatment with 17beta-estradiol, the decrease in the expression of alpha3 and beta1 without a change in Na+/K+-ATPase activity suggests that some isoform other than beta1 exist in rat uteri. The positive correlation between the reduction of alpha3-and the decrease of contraction frequency suggests the involvement of alpha3 isoform in uterine oscillation.     

On the molecular basis of ion permeation in the epithelial Na+ channel.

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two alpha, one beta, and one gamma homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of beta and gamma subunits, which precedes the second putative transmembrane alpha helix (M2). A residue in the alpha subunit (alphaS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of alpha, beta, and gamma subunits (alphaG587, betaG529, gammaS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of betaG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported alphaS589 mutations. We conclude that the residues alphaG587 to alphaS589 and homologous residues in the beta and gamma subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.     

Beta 1 subunits of sodium channels. Studies with subunit-specific antibodies

The purified Na+ channel from rat brain consists of alpha (260 kDa), beta 1 (36 kDa), and beta 2 (33 kDa) subunits. Pure beta 1 subunits were prepared from purified rat brain Na+ channels which had been adsorbed to hydroxylapatite resin and used to prepare specific anti-beta 1 subunit antiserum. Antibodies purified from this antiserum by antigen affinity chromatography immunoprecipitate 125I-labeled, purified beta 1 subunits and purified Na+ channels (measured as high affinity [3H] saxitoxin binding sites) and recognize beta 1 subunits on immunoblots of solubilized rat brain membranes. The affinity-purified anti-beta 1 antibodies recognize beta 1 subunits in rat spinal cord, heart, skeletal muscle, and sciatic nerve, but do not detect immunoreactive beta 1 subunits in eel electroplax or eel brain. The developmental time course of expression of immunoreactive beta 1 subunits in rat forebrain was measured by immunoprecipitation followed by immunoblotting with affinity-purified anti-beta 1 antibodies. The amount of immunoreactive beta 1 subunits increased steadily to adult levels during the first 21 days of postnatal development.     

A new subunit of the epithelial Na+ channel identifies regions involved in Na+ self-inhibition

The epithelial Na+ channel (ENaC) is the apical entry pathway for Na+ in many Na+-reabsorbing epithelia. ENaC is a heterotetrameric protein composed of homologous alpha, beta, and gamma subunits. Mutations in ENaC cause severe hypertension or salt wasting in humans; and consequently, ENaC activity is tightly controlled. According to the concept of Na+ self-inhibition, the extracellular Na+ ion itself can reduce ENaC activity. The molecular basis for Na+ self-inhibition is unknown. Here, we describe cloning of a new ENaC subunit from Xenopus laevis (epsilonxENaC). epsilonxENaC can replace alphaxENaC and formed functional, highly selective, amiloride-sensitive Na+ channels when coexpressed with betaxENaC and gammaxENaC. Channels containing epsilonxENaC showed strong inhibition by extracellular Na+. This Na+ self-inhibition was significantly slower than for alphaxENaC-containing channels. Using site-directed mutagenesis, we show that the proximal part of the large extracellular domain controls the speed of self-inhibition. This suggests that this region is involved in conformational changes during Na+ self-inhibition.     

Expression of Ca(2+) channel subunits during cardiac ontogeny in mice and rats: identification of fetal alpha(1C) and beta subunit isoforms

Functional cardiac L-type calcium channels are composed of the pore-forming alpha(1C) subunit and the regulatory beta(2) and alpha(2)/delta subunits. To investigate possible developmental changes in calcium channel composition, we examined the temporal expression pattern of alpha(1C) and beta(2) subunits during cardiac ontogeny in mice and rats, using sequence-specific antibodies. Fetal and neonatal hearts showed two size forms of alpha(1C) with 250 and 220 kDa. Quantitative immunoblotting revealed that the rat cardiac 250-kDa alpha(1C) subunit increased about 10-fold from fetal days 12-20 and declined during postnatal maturation, while the 220-kDa alpha(1C) decreased to undetectable levels. The expression profile of the 85-kDa beta(2) subunit was completely different: beta(2) was not detected at fetal day 12, rose in the neonatal stage, and persisted during maturation. Additional beta(2)-stained bands of 100 and 90 kDa were detected in fetal and newborn hearts, suggesting the transient expression of beta(2) subunit variants. Furthermore, two fetal proteins with beta(4) immunoreactivity were identified in rat hearts that declined during prenatal development. In the fetal rat heart, beta(4) gene expression was confirmed by RT-PCR. Cardiac and brain beta(4) mRNA shared the 3 prime region, predicting identical primary sequences between amino acid residues 62-519, diverging however, at the 5 prime portion. The data indicate differential developmental changes in the expression of Ca(2+) channel subunits and suggest a role of fetal alpha(1C) and beta isoforms in the assembly of Ca(2+) channels in immature cardiomyocytes.     

Modulation of cardiac Ca2+ channels in Xenopus oocytes by protein kinase C.

L-Type calcium channel was expressed in Xenopus laevis oocytes injected with RNAs coding for different cardiac Ca2+ channel subunits, or with total heart RNA. The effects of activation of protein kinase C (PKC) by the phorbol ester PMA (4 beta-phorbol 12-myristate 13-acetate) were studied. Currents through channels composed of the main (alpha 1) subunit alone were initially increased and then decreased by PMA. A similar biphasic modulation was observed when the alpha 1 subunit was expressed in combination with alpha 2/delta, beta and/or gamma subunits, and when the channels were expressed following injection of total rat heart RNA. No effects on the voltage dependence of activation were observed. The effects of PMA were blocked by staurosporine, a protein kinase inhibitor. beta subunit moderate the enhancement caused by PMA. We conclude that both enhancement and inhibition of cardiac L-type Ca2+ currents by PKC are mediated via an effect on the alpha 1 subunit, while the beta subunit may play a mild modulatory role.     

Fluorescence resonance energy transfer analysis of subunit stoichiometry of the epithelial Na+ channel

Activity of the epithelial Na(+) channel (ENaC) is rate-limiting for Na(+) (re)absorption across electrically tight epithelia. ENaC is a heteromeric channel comprised of three subunits, alpha, beta, and gamma, with each subunit contributing to the functional channel pore. The subunit stoichiometry of ENaC remains uncertain with electrophysiology and biochemical experiments supporting both a tetramer with a 2alpha:1beta:1gamma stoichiometry and a higher ordered channel with a 3alpha:3beta:3gamma stoichiometry. Here we used an independent biophysical approach based upon fluorescence resonance energy transfer (FRET) between differentially fluorophore-tagged ENaC subunits to determine the subunit composition of mouse ENaC functionally reconstituted in Chinese hamster ovary and COS-7 cells. We found that when all three subunits were co-expressed, ENaC contained at least two of each type of subunit. Findings showing that ENaC subunits interact with similar subunits in immunoprecipitation studies are consistent with these FRET results. Upon native polyacrylamide gel electrophoresis, moreover, oligomerized ENaC runs predominantly as a single species with a molecular mass of >600 kDa. Because single ENaC subunits have a molecular mass of approximately 90 kDa, these results also agree with the FRET results. The current results as a whole, thus, are most consistent with a higher ordered channel possibly with a 3alpha:3beta:3gamma stoichiometry.     

Antisera specific for the alpha 1, alpha 2, alpha 3, and beta subunits of the Na,K-ATPase: differential expression of alpha and beta subunits in rat tissue membranes

We have developed a panel of antibodies specific for the alpha 1, alpha 2, alpha 3, and beta subunits of the rat Na,K-ATPase. TrpE-alpha subunit isoform fusion proteins were used to generate three antisera, each of which reacted specifically with a distinct alpha subunit isotype. Western blot analysis of rat tissue microsomes revealed that alpha 1 subunits were expressed in all tissues while alpha 2 subunits were expressed in brain, heart, and lung. The alpha 3 subunit, a protein whose existence had been inferred from cDNA cloning, was expressed primarily in brain and copurified with ouabain-inhibitable Na,K-ATPase activity. An antiserum specific for the rat Na,K-ATPase beta subunit was generated from a TrpE-beta subunit fusion protein. Western blot analysis showed that beta subunits were present in kidney, brain, and heart. However, no beta subunits were detected in liver, lung, spleen, thymus, or lactating mammary gland. The distinct tissue distributions of alpha and beta subunits suggest that different members of the Na,K-ATPase family may have specialized functions.